The repair and regeneration of damaged or resected bone are problematic. Bone autografts show optimal skeletal incorporation, but often bring about complications. Hence, there is increasing interest in designing new biomaterials that could potentially be used in the form of scaffolds as bone substitutes. In this study we used a hydrophobic cross-linked polyurethane in a typical tissue-engineering approach, that is, the seeding and in vitro culturing of cells within a porous scaffold. The polyurethane porous scaffold had an average pore diameter of 624 microm. Using a perfusion bioreactor, we investigated the effect of shear stress on SAOS-2 human osteoblast proliferation and calcified matrix production. The physical, morphological, and compressive properties of the polyurethane foam were characterized. At a scaffold perfusion rate of 3 mL/min, in comparison with static conditions without perfusion, we observed 33% higher cell proliferation; higher secretion of osteopontin, osteocalcin, decorin, and type I collagen (9.16-fold, 71.9-fold, 30.6-fold, and 18.12-fold, respectively); and 10-fold increased calcium deposition. The design of the bioreactor and the design of the polyurethane foam aimed at obtaining cell colonization and calcified matrix deposition. This cultured biomaterial could be used, in clinical applications, as an osteoinductive implant for bone repair.